The Phylogenetic Relationship Revealed Three New Wood-Inhabiting Fungal Species from Genus Trechispora

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ORIGINAL RESEARCH published: 17 March 2021 doi: 10.3389/fmicb.2021.650195

The Phylogenetic Relationship Revealed Three New Wood-Inhabiting Fungal Species From Genus Trechispora

Wei Zhao1,2 and Chang-Lin Zhao1,2*

1 Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, China, 2 College of Biodiversity Conservation, Southwest Forestry University, Kunming, China

Wood-inhabiting fungi play a significant role in wood degradation and the cycle of matter in the ecological system. In the present study, three new wood-inhabiting fungal species, Trechispora bambusicola, Trechispora fimbriata, and Trechispora fissurata spp. nov., are Edited by: Dhanushka Nadeeshan nested in Trechispora, which are proposed based on a combination of morphological Wanasinghe, features and molecular evidence. Sequences of internal transcribed spacer (ITS) Key Laboratory for Plant Diversity and Biogeography of East Asia, and large subunit (nLSU) regions of the studied samples were generated, and the Kunming Institute of Botany, Chinese phylogenetic analyses were performed with maximum likelihood, maximum parsimony, Academy of Sciences, China and Bayesian inference methods. The phylogenetic analyses inferred from ITS showed Reviewed by: that T. bambusicola was sister to Trechispora stevensonii, T. fimbriata grouped Baokai Cui, Beijing Forestry University, China with Trechispora nivea, and T. fissurata grouped with Trechispora echinospora. The Yu-Cheng Dai, phylogenetic tree based on ITS + nLSU sequences demonstrated that T. bambusicola Beijing Forestry University, China formed a single lineage and then grouped with Trechispora rigida and T. stevensonii. *Correspondence: Chang-Lin Zhao T. fimbriata was sister to T. nivea. T. fissurata grouped with Trechispora thelephora. [email protected] Keywords: Hydnodontaceae, phylogeny, taxonomy, wood-inhabiting fungi, Yunnan Province

Specialty section: This article was submitted to Evolutionary and Genomic INTRODUCTION Microbiology, a section of the journal Trechispora P. Karst. (Hydnodontaceae, Trechisporales) was typiﬁed with Trechispora onusta Frontiers in Microbiology P. Karst. (Karsten, 1890). It is characterized by the resupinate to eﬀused basidiomata with Received: 08 January 2021 smooth to hydnoid to poroid hymenophore, a monomitic or dimitic hyphal structure Accepted: 01 February 2021 with clamped generative hyphae having typical ampullaceous septa, and short cylindric Published: 17 March 2021 Citation: Zhao W and Zhao C-L (2021) The Abbreviations: ITS, internal transcribed spacer; nLSU, large subunit; SWFC, herbarium of Southwest Forestry University, Kunming, China; KOH, 5% potassium hydroxide; CB, Cotton Blue; CB−, acyanophilous; IKI, Melzer’s reagent; IKI−, both Phylogenetic Relationship Revealed inamyloid and indextrinoid; L, mean spore length (arithmetic average for all spores); W, mean spore width (arithmetic Three New Wood-Inhabiting Fungal average for all spores); Q, variation in the L/W ratios between The studied specimens, n (a/b), number of spores (a) measured Species From Genus Trechispora. from given number (b) of specimens, spore measurements do not include ornamentation; CTAB, cetyltrimethylammonium Front. Microbiol. 12:650195. bromide; DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; MP, maximum parsimony; ML, maximum doi: 10.3389/fmicb.2021.650195 likelihood; BI, Bayesian inference; TBR, tree-bisection reconnection.

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basidia and smooth to verrucose or aculeate basidiospores based on field notes. Color terms follow Petersen(1996). (Karsten, 1890; Bernicchia and Gorjón, 2010). About 49 species Micromorphological data were obtained from the dried are currently known in Trechispora worldwide (Liberta, 1966, specimens and observed under a light microscope following 1973; Larsson, 1994, 1995, 1996; Ryvarden, 2002; Trichiès and Dai(2012). The following abbreviations were used: KOH = 5% Schultheis, 2002; Miettinen and Larsson, 2006; Ordynets et al., potassium hydroxide, CB = Cotton Blue, CB− = acyanophilous, 2015; Xu et al., 2019) and Index Fungorum1 and MycoBank2. IKI = Melzer’s reagent, IKI− = both inamyloid and indextrinoid, Larsson(2007) addressed the classification of corticioid fungi L = mean spore length (arithmetic average for all spores), and revealed that Trechispora farinacea (Pers.) Liberta grouped W = mean spore width (arithmetic average for all spores), with Trechispora hymenocystis (Berk. and Broome) K.H. Larss., Q = variation in the L/W ratios between the studied specimens, n in which both species nested within the family Hydnodontaceae (a/b) = number of spores (a) measured from given number (b) of Jülich. Based on the large subunit nuclear ribosomal RNA gene specimens, spore measurements do not include ornamentation. (nLSU) datasets, Albee-Scott and Kropp(2010) supported to transfer Hydnodon thelephorus (Lév.) Banker to Trechispora as Molecular Phylogeny Trechispora thelephora (Lév.) Ryvarden. The order Trechisporales Cetyltrimethylammonium bromide (CTAB) rapid plant genome was studied employing the internal transcribed spacer (ITS) extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, and nLSU regions, in which it suggested that Porpomyces China) was used to obtain genomic deoxyribonucleic acid Jülich, Sistotremastrum J. Erikss., Subulicystidium Parmasto, (DNA) from dried specimens, according to the manufacturer’s and Trechispora belonged to a highly supported clade and instructions following Zhao and Wu(2017). ITS region was Trechispora belongs to Hydnodontaceae and was closely related amplified with primer pair ITS5 and ITS4 (White et al., 1990). to Brevicellicium K.H. Larss. and Hjortstam (Telleria et al., Nuclear LSU region was amplified with primer pair LR0R and 2013). A phylogenetic study of Trechispora was addressed and LR73. The polymerase chain reaction (PCR) procedures for ITS demonstrated that Trechispora cyatheae Ordynets, Langer and and nLSU following Zhao and Wu(2017). The PCR products K.H. Larss. and Trechispora echinocristallina Ordynets, Langer were purified and directly sequenced at Kunming Tsingke and K.H. Larss. clustered into Trechispora as new members, Biological Technology Limited Company, Kunming, Yunnan inferred from the combined data of the ITS and LSU datasets Province, China. All newly generated sequences were deposited (Ordynets et al., 2015). The phylogeny of Trechisporales was at GenBank (Table 1). inferred from a combined dataset of ITS-nLSU sequences and Sequencher 4.6 (GeneCodes, Ann Arbor, United States) was showed that Porpomyces, Scytinopogon Singer, and Trechispora used to edit the DNA sequence. Sequences were aligned in grouped together and nested within family Hydnodontaceae (Liu MAFFT 74 using the “G-INS-I” strategy and manually adjusted et al., 2019). Phylogram generated from analysis of ITS sequence in BioEdit (Hall, 1999). The sequence alignment was deposited dataset of Trechispora showed that Trechispora echinospora in TreeBase (submission ID 25879). Sequences of Fibrodontia Telleria was sister to the clade formed by Trechispora araneosa alba Yurchenko and Sheng H. Wu and Fibrodontia gossypina (Hohn. and Litsch.) K.H. Larss., T. farinacea, T. hymenocystis, Parmasto retrieved from GenBank were used as an outgroup in and Trechispora mollusca (Pers.) Liberta with a low support the ITS + nLSU analyses by following Ordynets et al.(2015). (Phookamsak et al., 2019). The ITS + nLSU dataset comprised Maximum parsimony (MP) analyses were applied to the 22 species and revealed that Trechispora yunnanensis C.L. Zhao ITS + nLSU dataset sequences. Approaches to phylogenetic formed a monophyletic lineage within Trechispora and was analysis followed Zhao and Wu(2017), and the tree construction closely related to Trechispora byssinella (Bourdot) Liberta and procedure was performed in PAUP∗ version 4.0b10 (Swofford, Trechispora laevis K.H. Larss. (Xu et al., 2019). 2002). All characters were equally weighted and gaps were During the studies on wood-inhabiting fungi in southern treated as missing data. Trees were inferred using the heuristic China, three species of Trechispora could not be assigned search option with tree-bisection reconnection (TBR) branch to any described species. Obtaining sequences from the new swapping and 1000 random sequence additions. Max-trees were taxa, the authors examine taxonomy and phylogeny of three set to 5000, branches of zero length were collapsed, and all new species within the genus Trechispora, based on the ITS parsimonious trees were saved. Clade robustness was assessed and nLSU sequences. using a bootstrap (BT) analysis with 1000 replicates (Felsenstein, 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), MATERIALS AND METHODS and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree generated. Datamatrix was also analyzed Morphology using maximum likelihood (ML) approach with RAxML-HPC2 The studied specimens are deposited at the herbarium of through the Cipres Science Gateway with GTR + I + G molecular Southwest Forestry University (SWFC), Kunming, Yunnan evolution model5 (Miller et al., 2009). Branch support (BS) for Province, China. Macromorphological descriptions were ML analysis was determined by 1000 BT replicates.

3 1http://www.indexfungorum.org/Names/Names.asp http://lutzonilab.org/nuclear-ribosomal-dna/ 4 2https://www.mycobank.org/Biolomics.aspx?Table=Mycobank&Page=200& http://maﬀt.cbrc.jp/alignment/server/ ViewMode=Basic 5www.phylo.org

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TABLE 1 | List of species, specimens, and GenBank accession numbers of sequences used in this study.

Species name Sample no. GenBank accession no. References

ITS nLSU

Fibrodontia alba TNMF 24944 KC928274 KC928275 Yurchenko and Wu(2014) Fibrodontia gossypina GEL 5042 DQ249274 AY646100 Unpublished Trechispora araneosa KHL 8570 AF347084 AF347084 Larsson et al.(2004) Trechispora bambusicola CLZhao 3302 MW544021 MW520171 This study Trechispora bambusicola CLZhao 3305 MW544022 MW520172 This study Trechispora bispora CBS 142.63 MH858241 MH869842 Vu et al.(2019) Trechispora byssinella UC 2023068 KP814481 – Unpublished Trechispora cohaerens TU 110332 UDB008249 – Ordynets et al.(2015) Trechispora cohaerens TU 115568 UDB016421 – Ordynets et al.(2015) Trechispora confinis KHL 11064 AF347081 AF347081 Larsson et al.(2004) Trechispora cyatheae FR-0219442 UDB024014 UDB024014 Ordynets et al.(2015) Trechispora cyatheae FR-0219443 UDB024015 UDB024015 Ordynets et al.(2015) Trechispora echinocristallina FR-0219445 UDB024018 UDB024018 Ordynets et al.(2015) Trechispora echinocristallina FR-0219448 UDB024022 UDB024022 Ordynets et al.(2015) Trechispora echinospora E11/37-03 JX392845 JX392846 Telleria et al.(2013) Trechispora echinospora E09/60-06 JX392847 JX392848 Telleria et al.(2013) Trechispora echinospora E11/37-05 – JX392849 Telleria et al.(2013) Trechispora farinacea KHL 8451 AF347082 AF347082 Unpublished Trechispora farinacea KHL 8793 AF347089 AF347089 Larsson et al.(2004) Trechispora fissurata CLZhao 995 MW544026 MW520176 This study Trechispora fissurata CLZhao 4571 MW544027 MW520177 This study Trechispora fimbriata CLZhao 4154 MW544023 MW520173 This study Trechispora fimbriata CLZhao 7969 MW544024 MW520174 This study Trechispora fimbriata CLZhao 9006 MW544025 MW520175 This study Trechispora hymenocystis KHL 8795 AF347090 AF347090 Unpublished Trechispora hymenocystis TL 11112 UDB000778 UDB000778 Ordynets et al.(2015) Trechispora incisa EH 24/98 AF347085 – Unpublished Trechispora kavinioides KGN 981002 AF347086 AF347086 Larsson et al.(2004) Trechispora laevis TU 115551 UDB016468 – Ordynets et al.(2015) Trechispora mollusca DLL 2010-077 JQ673209 – Ordynets et al.(2015) Trechispora mollusca DLL 2011-186 KJ140681 – Ordynets et al.(2015) Trechispora nivea MA-Fungi 76238 JX392824 JX392825 Telleria et al.(2013) Trechispora nivea MA-Fungi 76257 JX392826 JX392827 Telleria et al.(2013) Trechispora nivea MA-Fungi 82480 JX392829 JX392830 Telleria et al.(2013) Trechispora nivea MA-Fungi 74044 JX392832 JX392833 Telleria et al.(2013) Trechispora regularis KHL 10881 AF347087 AF347087 Larsson et al.(2004) Trechispora rigida URM 85754 – MH279999 Unpublished Trechispora stevensonii MA-Fungi 70669 JX392841 JX392842 Telleria et al.(2013) Trechispora stevensonii HJM 18087 – MH290761 Unpublished Trechispora stevensonii KHL 14654 – MH290762 Unpublished Trechispora stevensonii TU 115499 UDB016467 UDB016467 Ordynets et al.(2015) Trechispora stellulata UC 2022880 KP814437 – Unpublished Trechispora stellulata UC 2023099 KP814451 – Unpublished Trechispora subsphaerospora KHL 8511 AF347080 AF347080 Larsson et al.(2004) Trechispora thelephora URM 85757 – MH280001 Unpublished Trechispora thelephora URM 85758 – MH280002 Unpublished Trechispora yunnanensis CLZhao 210 MN654918 MN654921 Xu et al.(2019) Trechispora yunnanensis CLZhao 214 MN654919 MN654922 Xu et al.(2019) Trechispora yunnanensis CLZhao 215 MN654920 MN654923 Xu et al.(2019)

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FIGURE 1 | Maximum Parsimony strict consensus tree illustrating the phylogeny of three new species and related species in Trechispora based on ITS sequences. Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap proportion values > 50%, and Bayesian posterior probabilities > 0.95, respectively.

MrModeltest 2.3 (Nylander, 2004) was used to determine values > 75%, MP BT values > 75%, or Bayesian posterior the best-fit evolution model (GTR + I + G) for each data probabilities (PP) > 0.95. set for Bayesian inference (BI) of the phylogeny. BI was calculated with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Four Markov chains were run for two runs from RESULTS random starting trees for 1 million generations and trees were sampled every 100 generations; the first one-fourth of Molecular Phylogeny generations were discarded as burn-in. A majority rule consensus In the ITS dataset, the sequences from 43 fungal specimens tree of all remaining trees was calculated. Branches were representing 25 species were included. The dataset had an considered as significantly supported if they received ML BT aligned length of 1034 characters, of which 521 characters

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FIGURE 2 | Maximum Parsimony strict consensus tree illustrating the phylogeny of three new species and related species in Trechispora based on ITS + nLSU sequences. Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap proportion values > 50%, and Bayesian posterior probabilities > 0.95, respectively.

are constant, 86 are variable and parsimony-uninformative, In the ITS + nLSU dataset, it included sequences from 49 and 427 are parsimony-informative. MP analysis yielded fungal specimens representing 27 species. The dataset had an 26 equally parsimonious trees (TL = 2048, CI = 0.4561, aligned length of 2256 characters, of which 1387 characters are HI = 0.5439, RI = 0.6174, RC = 0.2816). Best model for constant, 188 are variable and parsimony-uninformative, and the ITS dataset estimated and applied in the Bayesian 681 are parsimony-informative. MP analysis yielded 100 equally analysis: GTR + I + G, lset nst = 6, rates = invgamma; parsimonious trees (TL = 2811, CI = 0.4963, HI = 0.5037, prset statefreqpr = dirichlet (1,1,1,1). Bayesian analysis RI = 0.6409, RC = 0.3180). Best model for the ITS dataset and ML analysis resulted in a similar topology to MP estimated and applied in the Bayesian analysis: GTR + I + G, analysis, with an average standard deviation of split lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet frequencies = 0.009985 (BI). (1,1,1,1). Bayesian analysis and ML analysis resulted in a similar The phylogeny (Figure 1) inferred from ITS sequences topology to MP analysis, with an average standard deviation of showed that Trechispora bambusicola was sister to Trechispora split frequencies = 0.009991 (BI). stevensonii (Berk. and Broome) K.H. Larss, and Trechispora The phylogenetic tree (Figure 2) inferred from ITS + nLSU ﬁmbriata grouped with Trechispora nivea. T. ﬁssurata sequences demonstrated 27 species of Trechispora and revealed grouped with T. echinospora Telleria, M. Dueñas, I. Melo, that T. bambusicola formed a single lineage and then grouped and M.P. Martín. with Trechispora rigida (Berk.) K.H. Larss. and T. stevensonii.

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Hyphal structure Monomitic, generative hyphae with clamp connections, hyaline, thick-walled, up to 0.7 µm, richly branched, 2–3 µm in diameter, IKI−, CB−; hyphae unchanged in KOH. Hymenium Cystidia and cystidioles absent; basidia shortly cylindrical to subclavate with median constriction, with 4-sterigmata and a basal clamp connection, 9–13 × 2.5–5 µm, basidioles dominant, in shape similar to basidia, but slightly smaller. Basidiospores Ellipsoid, hyaline, thick-walled, ornamented, sparse aculei, sharp, IKI−, CB−, (2.6−)2.9–3.5(−3.9) × 2–2.7 µm, L = 3.18 µm, W = 2.41 µm, Q = 1.26–1.38 (n = 60/2). Type of rot White rot. Additional specimen examined CHINA, Yunnan Province, Pu’er, Laiyanghe National Forestry Park, on dead bamboo, 30 September 2017, CLZhao 3302 (SWFC).

Trechispora fimbriata C.L. Zhao, sp. nov. MycoBank no.: MB 838613 (Figures 5, 6). Holotype—China, Yunnan Province, Puer, Jingdong County, Wuliangshan National Nature Reserve, on the angiosperm trunk, October 5, 2017, CLZhao 4154 (SWFC). Etymology—Fimbriata (Lat.): refers to the fimbriate margin of the basidiomata. Basidiomata FIGURE 3 | Basidiomata of Trechispora bambusicola (holotype): Bars: Annual, adnate, without odor or taste when fresh, becoming (A) 2 cm; (B) 1 mm. fragile upon drying, up to 10 cm long and 3 cm wide, 100–200 µm thick. Hymenial surface hydnoid, with aculei, cylindrical, blunt, 0.4–0.7 mm long, white to pink when fresh, turn to pink to buff T. fimbriata was sister to T. nivea. T. fissurata grouped with upon drying. Margin white to cream, thinning out, fimbriate. T. thelephora (Lév.) Ryvarden. Hyphal system Monomitic, generative hyphae with clamp connections, hyaline, Taxonomy thick-walled, up to 0.6 µm, branched, 2–4 µm in diameter, IKI−, Trechispora bambusicola C.L. Zhao, sp. nov. CB−; hyphae unchanged in KOH. MycoBank no.: MB 838612 (Figures 3, 4). Holotype—China, Yunnan Province, Pu’er, Laiyanghe Hymenium National Forest Park, on dead bamboo, 30 September 2017, Cystidia and cystidioles absent; basidia shortly cylindrical CLZhao 3305 (SWFC). with median constriction, with 4–6 sterigmata and a × Etymology—Bambusicola (Lat.): referring to occurrence basal clamp connection, 7–11.5 3.5–5 µm, basidioles on bamboo stump. dominant, in shape similar to basidia, but slightly smaller. Basidiomata Basidiospores Annual, adnate, soft, and fragile, without odor or taste when Ellipsoid, hyaline, thick-walled, ornamented, sparse aculei, sharp, fresh, becoming granulose upon drying, up to 15 cm long and IKI−, CB−, (2.5−)3–3.6(−3.8) × 2.4–3.2 µm, L = 3.25 µm, 5 cm wide, 50–300 µm thick. Hymenial surface odontioid, W = 2.63 µm, Q = 1.17–1.38 (n = 90/3). aculei cylindrical to conical, blunt, 0.3–0.5 mm long, white to cream when fresh, turn to cream to buff upon drying. Type of rot Margin white to cream. White rot.

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FIGURE 4 | Microscopic structures of Trechispora bambusicola (drawn from the holotype): (A) Section of hymenium. (B) Basidiospores. (C) Basidia and basidioles. Bars: (A,C) 10 µm; (B) 5 µm.

Additional specimens examined 2018, CLZhao 7969 (SWFC); on angiosperm trunk, October 15, China, Yunnan Province, Yuxi, Xinping County, Mopanshan 2018, CLZhao 9006 (SWFC). National Forestry Park, on living tree of angiosperm, August 9, Trechispora ﬁssurata C.L. Zhao, sp. nov. MycoBank no.: MB 838614 (Figures 7, 8). Holotype—China, Yunnan Province, Puer, Jingdong County, Wuliangshan National Nature Reserve, on angiosperm trunk, October 6, 2017, CLZhao 4571 (SWFC). Etymology—Fissurata (Lat.): refers to the cracking ﬁssures on hymenial surface.

Basidiomata Annual, adnate, without odor or taste when fresh, becoming cracking upon drying, up to 8 cm long and 4.5 cm wide, 400–800 µm thick. Hymenial surface hydnoid, with aculei, cylindrical to conical, sharp, 0.5–0.9 mm long, cream to straw yellow when fresh, turn to cream to yellow upon drying. Margin cream to yellow.

Hyphal system Monomitic, generative hyphae with clamp connections, hyaline, thick-walled, up to 0.8 µm, branched, 2.5–5 µm in diameter, IKI−, CB−; hyphae unchanged in KOH.

Hymenium Cystidia and cystidioles absent; basidia shortly clavate to tubular, with 4-sterigmata and a basal clamp connection, 8–10.5 × 2.5– FIGURE 5 | Basidiomata of Trechispora ﬁmbriata (holotype). Bars: (A) 5 mm; (B) 1 mm. 4.5 µm, basidioles dominant, in shape similar to basidia, but slightly smaller.

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FIGURE 6 | Microscopic structures of Trechispora ﬁmbriata (drawn from the holotype). (A) Basidiospores. (B) Basidia and basidioles. (C) Section of hymenium. Bars: (A) 5 µm; (B,C) 10 µm.

Basidiospores genus, in which it implied the phylogenetic relationship among Ellipsoid, hyaline, thick-walled, ornamented, dense aculei, sharp, species of Trechispora. In the present study, based on the ITS IKI−, CB−, (3−)3.3–4(−4.3) × (2.5−)2.8–3.5(−3.9) µm, sequences (Figure 1), T. bambusicola was sister to T. stevensonii L = 3.67 µm, W = 3.19 µm, Q = 1.13–1.17 (n = 60/2). (Berk. and Broome) K.H. Larss; T. fimbriata grouped with T. nivea; T. fissurata grouped with T. echinospora Telleria, Type of rot M. Dueñas, I. Melo, and M.P. Martín. Further ITS + nLSU White rot. dataset (Figure 2) revealed that T. bambusicola formed a single Additional specimen examined lineage and then grouped with T. rigida and T. stevensonii; CHINA, Yunnan Province, Yuxi, Xinping County, Mopanshan T. fimbriata was sister to T. nivea; T. fissurata grouped with Forestry Park, on the fallen angiosperm branch, January 17, 2017, T. thelephora. However, T. rigida differs in its dirty white to CLZhao 995 (SWFC). buff hymenophore (Larsson, 1996). T. stevensonii is separated from T. bambusicola by the smooth to hydnoid hymenophore and larger basidiospores (4–4.5 × 3–3.5 µm; Larsson, 1995). DISCUSSION T. nivea differs from T. fimbriata by the white to light ochraceous hymenial surface (Persoon, 1794). T. echinospora differs from Phylogenetically, Phookamsak et al.(2019) introduced the T. fissurata by the farinaceous to grandinioid hymenophore and phylogram generated from BI analysis of ITS sequence dataset larger, globose basidiospores (3.3–4 × 2.8–3.5 µm; Phookamsak of Trechispora sequences and included most taxa in this et al., 2019) and T. thelephora differs in its pileate to stipitate

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T. clancularis differs in the poroid to irpicoid hymenophore and slightly cyanophilous basidiospores (Larsson, 1994); T. stellulata differs in the arachnoid to byssoid hymenophore with whitish hymenial surface (Liberta, 1966); and T. subhelvetica differs in the narrower basidiospores (3–4 × 2–2.5 µm; Parmasto, 1965). Trechispora fissurata resembles several species with similar features of having the hydnoid hymenophore and a monomitic hyphal system: T. nivea (Pers.) K.H. Larss., T. stevensonii (Berk. and Broome) K.H. Larss., and Trechispora verruculosa (G. Cunn.) K.H. Larss., but T. nivea by the white to pale ochraceous hymenial surface and thin-walled generative hyphae encrusted with granular crystals (Bernicchia and Gorjón, 2010); T. stevensonii by the white to ochraceous hymenial surface and larger basidiospores (4–4.5 × 3–3.5 µm; Larsson, 1995); T. verruculosa by the slightly cyanophilous and larger basidiospores (4.5– 5.5 × 3.5–4.5 µm; Larsson, 1996). Currently, eight species of Trechispora have been reported from China (Dai, 2011; Xu et al., 2019), Trechispora alnicola, Trechispora cohaerens, T. farinacea, T. microspora, T. nivea, Trechispora polygonospora Ryvarden, Trechispora subsphaerospora (Litsch.) Liberta, and T. yunnanensis, and one species of T. yunnanensis was found in Yunnan Province of China and it differs from three new species by having a smooth to farinaceous hymenial surface and larger basidiospores (7– 8.5 × 5–5.5 µm; Xu et al., 2019). Three new taxa do not closely group together in phylogenetic trees, and morphologically, T. bambusicola differs from T. fimbriata and T. fissurata by having granulose basidiomata with cream to buff hymenial surface and growth on dead bamboo. T. fimbriata differs in its fimbriate margin of the basidiomata with pink to buff hymenial surface. In addition, the ectomycorrhizal fungi (EcM) play an important role in ecosystems based on their mutualistic association with many groups of plants (Heijden et al., FIGURE 7 | Basidiomata of Trechispora fissurata (holotype). Bars: (A) 1 cm; 2015). Vanegas-León et al.(2019) discovered the Trechisporales (B) 1 mm. basidiomes and root colonization from T. thelephora basidiome. In the present study, T. fissurata was sister to T. thelephora based on ITS + nLSU phylogenetic analysis (Figure 2), with light yellow brown surface and larger (4–5 × 3.4–4.5 µm; which implied that both species have close evolutionary Albee-Scott and Kropp, 2010). relationship. However, T. fissurata grows on deeply decayed In the present study, three new species, T. bambusicola, wood, and T. thelephora is a soil-inhabiting fungus. Therefore, T. fimbriata, and T. fissurata spp. nov. are found from future investigations in both inhabiting types are needed rotten wood. Morphologically, T. bambusicola is similar to to determine whether the natural selection or other factors T. cyatheae Ordynets, Langer and K.H. Larss. by sharing pushes the different direction on inhabiting soil/wood among the characteristics of soft and fragile basidiomata. However, Trechispora. T. cyatheae differs from T. bambusicola by having farinaceous In the habitat and distribution, Hibbett et al.(2014) revealed to grandinioid hymenophore and thin-walled generative hyphae that most species of Trechispora is considered as soil-inhabiting. (Ordynets et al., 2015). Later, some species were found on deeply decayed wood fungi Trechispora fimbriata has similar characteristics of having (Bernicchia and Gorjón, 2010; Dai, 2011). However, some the fimbriate margin with Trechispora canariensis Ryvarden species in Trechispora are a typical feature of ectomycorrhizal and Liberta, Trechispora clancularis (Park.-Rhodes) K.H. fungi as frequently forming basidiomes on soil (Dunham Larss., Trechispora microspora (P. Karst.) Liberta, Trechispora et al., 2007; Vanegas-León et al., 2019). In the neotropical stellulata (Bourdot and Galzin) Liberta, and Trechispora and subtropical region, the ectomycorrhizal basidiomes are subhelvetica (Parmasto) Liberta. However, T. canariensis found; however, the researches on the new taxa related to differs in its arachnoid to pelliculose hymenophore and larger wood-decaying fungi of Trechispora from China are poorly basidiospores (5–7 × 3–3.5 µm; Ryvarden and Liberta, 1978); reported. Further studies may focus on the relationships between

Frontiers in Microbiology| www.frontiersin.org 9 March 2021| Volume 12| Article 650195 fmicb-12-650195 March 11, 2021 Time: 17:8 # 10

Zhao and Zhao Three Potential New Ectomycorrhizal Species

FIGURE 8 | Microscopic structures of Trechispora ﬁssurata (drawn from the holotype). (A) Basidiospores. (B) Basidia and basidioles. (C) Section of hymenium. Bars: (A) 5 µm; (B,C) 10 µm.

the plants and species from Trechispora and try to better nLSU MW520171-MW520177) and MycoBank (MB 838612-MB understand the evolutionary directions between soil-inhabiting 838614). and decayed wood fungi of Trechispora; many fungal studies on phylogeny and application were from these areas, which will be useful to push future researches for the genus AUTHOR CONTRIBUTIONS Trechispora (Dai, 2011; Cui et al., 2019; Shen et al., 2019; C-LZ collected the species. WZ performed the molecular Zhu et al., 2019; Richter et al., 2019; Angelini et al., 2020; phylogenetic analyses. Both authors were responsible for the Bao et al., 2020). morphological analysis and description of the collections, planned, organized, and evaluated critically the experimental parts, wrote the manuscript, contributed to the article, and DISCLOSURE approved the submitted version. All the experiments undertaken in this study comply with the current laws of the People’s Republic of China. FUNDING

The research was supported by the Yunnan Fundamental DATA AVAILABILITY STATEMENT Research Project (Grant No. 202001AS070043), the Key Laboratory of Forest Resources Conservation and Utilization The data presented in the study are deposited in in the Southwest Mountains of China Ministry of Education, the https://www.ncbi.nlm.nih.gov/GenBank and https: Southwest Forestry University (KLESWFU-202003), and //www.mycobank.org/page/Home/MycoBank repository the High-level Talents Program of Yunnan Province accession number of GenBank (ITS MW544021-MW544027 and (YNQR-QNRC-2018-111).

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Zhao and Zhao Three Potential New Ectomycorrhizal Species

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Frontiers in Microbiology| www.frontiersin.org 11 March 2021| Volume 12| Article 650195